Sex-dependent effects of acute stress in adolescence or adulthood on appetitive motivation.

RATIONALE: Intensely stressful experiences can lead to long-lasting changes in appetitive and aversive behaviors. In humans, post-traumatic stress disorder increases the risk of comorbid appetitive disorders including addiction and obesity. We have previously shown that an acute stressful experience in adult male rats suppresses motivation for natural reward. OBJECTIVES: We examine the impact of sex and age on the effects of intense stress on action-based (instrumental) and stimulus-based (Pavlovian) motivation for natural reward (food). METHODS: Rats received 15 unsignaled footshocks (stress) in a single session followed by appetitive training and testing in a distinct context. In Experiment 1, stress occurred in either adolescence (PN28) or adulthood (PN70) with appetitive training and testing beginning on PN71 for all rats. In Experiment 2, stress and appetitive training/testing occurred in adolescence. RESULTS: Acute stress in adolescent females suppressed instrumental motivation assessed with progressive ratio testing when testing occurred in late adolescence or in adulthood, whereas in males stress in adolescence did not suppress instrumental motivation. Acute stress in adulthood did not alter instrumental motivation. In contrast, Pavlovian motivation assessed with single-outcome Pavlovian-to-instrumental transfer (SO-PIT) was consistently enhanced in females following adolescent or adult stress. In males, however, stress in adolescence had no effect, whereas stress in adulthood attenuated SO-PIT. CONCLUSIONS: Acute stress in adolescence or adulthood altered instrumental motivation and stimulus-triggered Pavlovian motivation in a sex and developmentally specific manner. These findings suggest that the persistent effects of acute stress on Pavlovian and instrumental motivational processes differ in females and males, and that males may be less vulnerable to the deleterious effects of intense stress during adolescence on appetitive motivation.

Role of nucleus accumbens D1-type medium spiny neurons in the expression and extinction of sign-tracking.

While sign-tracking, also known as autoshaping, has been studied for many decades, only recently has the tendency to show sign-tracking behavior been linked to the development and persistence of addiction. Sign-tracking is dependent upon dopamine activity in the nucleus accumbens (NAc). The NAc is comprised predominantly of medium spiny projection neurons (MSN) that can be differentiated by their D1-like or D2-like dopamine receptor expression. Here we determined how reducing activity of D1-type MSNs in the NAc affects the expression and extinction of sign-tracking. To address this, we transfected the NAc of transgenic male and female rats that selectively express Cre recombinase in D1-type MSNs with a DIO viral vector expressing hM4Di. Cre- rats were given the same viral infusion but did not express the hM4Di receptor and therefore served as controls. Rats were then conditioned to associate lever presentations with pellet delivery. After sign-tracking was established, all rats were administered clozapine-n-oxide (CNO) prior to three additional conditioning sessions to assess the effects of NAc D1-MSNs inhibition on sign-tracking in the presence of reward. CNO treatment did not alter the expression of sign-tracking in Cre+ or Cre- rats. Next rats underwent extinction training where lever presentations occurred without pellet delivery and all rats received a CNO injection prior to each extinction session. In these extinction conditions, Cre+ rats exhibited robust extinction of sign-tracking across sessions, whereas Cre- rats did not. To determine if D1-MSN inhibition merely produced a temporary cessation of sign-tracking or instead had facilitated a persistent loss of sign-tracking, we evaluated the reemergence of sign-tracking in a test for reconditioning. During testing, reintroduction of the CS-US pairing did not promote the reemergence of sign-tracking in Cre+ rats, but restored sign-tracking in Cre- rats. Thus, chemogenetic inhibition of NAc D1-MSNs promoted extinction of sign-tracking. Collectively, these data suggest that D1-MSNs play an important role in resistance to extinction that typifies sign-tracking behavior.

Acute stress persistently alters instrumental motivation without affecting appetitive Pavlovian conditioning, extinction, or contextual renewal.

In two experiments, we adapted the stress-enhanced fear learning approach to evaluate the persistent effects of acute stress on appetitive learning and motivation in adult male Long Evans rats. In Experiment 1, we found that exposure to a battery of footshocks in one context had no effect on the acquisition, extinction, or contextual renewal of an appetitive Pavlovian discrimination in different contexts. However, when rats were subsequently trained to respond on a progressive ratio instrumental schedule, rats with a history of shock showed lower response rates and progressive ratio break points. Extinction of the shock-associated context had little effect on progressive ratio responding. In Experiment 2, we replicated this instrumental responding deficit with a continuous reinforcement schedule when the Pavlovian phases did not intervene in the time between shock and instrumental testing. Our findings here demonstrate that highly stressful acute experiences produce long-lasting deficits in instrumental motivation for food in male rats.

Persistent effects of acute trauma on Pavlovian-to-instrumental transfer.

In humans, an acutely traumatic experience can lead to post-traumatic stress disorder (PTSD), which is often characterized by changes in anxiety and motivation months after trauma. There are few demonstrations of the persistent motivational effects of an acute stressor in rodent approaches to PTSD. In two experiments, we evaluated the persistent effects of a battery of footshocks in one context on appetitive Pavlovian conditioning, instrumental learning, and Pavlovian-to-instrumental transfer (PIT) in a different context. In Experiment 1, a battery of footshocks before appetitive training caused deficits in single-outcome PIT (SO-PIT) in male Long Evans rats. The same battery of footshocks after appetitive training, but before testing had little effect on SO-PIT overall, but there were some deficits in within-stimulus expression of SO-PIT. In Experiment 2, the battery of footshocks had no effect on sensory-specific PIT in male or female rats, but two sex differences emerged: males showed more generalized fear from the aversive to the appetitive context compared to females, and females showed less evidence for sensory-specific PIT compared to males. Males showed robust sensory-specific PIT, with clear extinction and spontaneous recovery of the sensory-specific PIT effect across test sessions. These findings show that (a) an acute trauma can have persistent effects on general motivational processes and (b) in sensory-specific PIT, females may show transfer through generalized motivational processes, whereas males may rely on specific features of the cues and outcomes to augment instrumental responding selectively. We discuss implications for current approaches to stress and motivation in preclinical approaches to PTSD.

Effects of hM4Di activation in CamKII basolateral amygdala neurons and CNO treatment on sensory-specific vs. general PIT: refining PIT circuits and considerations for using CNO.

BACKGROUND: Pavlovian stimuli can influence instrumental behaviors via phenomena such as Pavlovian-to-instrumental transfer (PIT). PIT arises via dissociable processes as sensory-specific PIT (SS-PIT) and general PIT. The basolateral amygdala (BLA) mediates SS-PIT, but not general PIT. However, the specific BLA neuronal populations involved are unknown. AIMS: To determine the contribution of glutamatergic BLA neurons to the expression of SS-PIT and to the recall of sensory-specific properties of stimulus-outcome associations. METHODS: BLA neurons were transduced with virus containing either GFP or hM4Di, driven by the CamKII promoter. Rats were then tested for SS and general PIT and subsequently for expression of Pavlovian outcome devaluation effects and conditioned taste aversion following injections of vehicle or clozapine-N-oxide (CNO, the hM4Di agonist). RESULTS: CNO selectively blocked SS-PIT in the hM4Di-expressing group, but not controls, without altering expression of Pavlovian outcome devaluation or sensory-specific taste aversion in either group. Unexpectedly, CNO disrupted general PIT in both groups. CONCLUSIONS: CamKII BLA neurons mediate the expression of SS-PIT by enabling Pavlovian stimuli to trigger recall of the correct action-outcome associations rather than by mediating recall of the sensory-specific properties of the stimulus-outcome association. Separately, our data demonstrate that CNO alone is sufficient to disrupt affective, but not sensory-specific processes, an effect that was not due to generalized motor disruption. This non-specific effect on general PIT may be related to CNO-induced shifts in internal state. Together, these data identify BLA CamKII neurons as critical for the expression of SS-PIT and reveal important considerations for using CNO to study general affective motivation.

Affective Pavlovian motivation is enhanced in obesity susceptible populations: Implications for incentive motivation in obesity.

Continually rising global obesity rates present a major challenge to human health. The contribution of Pavlovian motivational processes to overeating and obesity has become increasingly apparent. In humans, brain and behavioral reactivity to food-related stimuli positively correlates with subsequent weight gain. In concordance with this, selectively bred obesity-prone rats show stronger single-outcome Pavlovian-to-instrumental transfer (SO PIT) than obesity-resistant rats, providing support for the hypothesis that enhanced Pavlovian motivation is a pre-existing phenotype of obesity-susceptibility. However, whether obesity-susceptibility in outbred rats is associated with similar enhancements in PIT was unknown. Moreover, given that SO PIT does not distinguish between sensory specific and general affective motivational processes, it was unclear which of these was linked to obesity-susceptibility. Thus, here we determined whether obesity-susceptibility is associated with enhanced Sensory Specific (SS) PIT versus General PIT using both outbred and selectively bred populations. Rats were trained with two action-outcome and three stimulus-outcome associations; two of the Pavlovian and instrumental associations shared a common outcome. During PIT testing, the influence of the Pavlovian stimuli on the two instrumental responses were measured simultaneously. In outbred rats, expression of General PIT was positively correlated with subsequently determined obesity-susceptibility. In selectively bred rats, General PIT was stronger in obesity-prone versus obesity-resistant rats. Jointly, these data show that enhanced affective Pavlovian motivation is tightly linked to obesity vulnerability, supporting a role for phenotypic differences in incentive motivation in vulnerability to obesity. This has important implications for obesity prevention and for the specific neurocircuitry underlying enhanced food-seeking in vulnerable populations.

Knock-In Rat Lines with Cre Recombinase at the Dopamine D1 and Adenosine 2a Receptor Loci.

Genetically modified mice have become standard tools in neuroscience research. Our understanding of the basal ganglia in particular has been greatly assisted by BAC mutants with selective transgene expression in striatal neurons forming the direct or indirect pathways. However, for more sophisticated behavioral tasks and larger intracranial implants, rat models are preferred. Furthermore, BAC lines can show variable expression patterns depending upon genomic insertion site. We therefore used CRISPR/Cas9 to generate two novel knock-in rat lines specifically encoding Cre recombinase immediately after the dopamine D1 receptor (Drd1a) or adenosine 2a receptor (Adora2a) loci. Here, we validate these lines using in situ hybridization and viral vector mediated transfection to demonstrate selective, functional Cre expression in the striatal direct and indirect pathways, respectively. We used whole-genome sequencing to confirm the lack of off-target effects and established that both rat lines have normal locomotor activity and learning in simple instrumental and Pavlovian tasks. We expect these new D1-Cre and A2a-Cre rat lines will be widely used to study both normal brain functions and neurological and psychiatric pathophysiology.

Sign-tracking is an expectancy-mediated behavior that relies on prediction error mechanisms.

When discrete localizable stimuli are used during appetitive Pavlovian conditioning, "sign-tracking" and "goal-tracking" responses emerge. Sign-tracking is observed when conditioned responding is directed toward the CS, whereas goal-tracking manifests as responding directed to the site of expected reward delivery. These behaviors seem to rely on distinct, though overlapping neural circuitries, and, possibly, distinct psychological processes as well, and are thought to be related to addiction vulnerability. One currently popular view is that sign-tracking reflects an incentive motivational process, whereas goal-tracking reflects the influence of more top-down cognitive processes. To test these ideas, we used illness-induced outcome-devaluation and Kamin blocking procedures to determine whether these behaviors rely on similar or distinct underlying associative mechanisms. In Experiments 1 and 2 we showed that outcome-devaluation reduced sign-tracking responses, demonstrating that sign-tracking is controlled by reward expectancies. We also observed that post-CS goal-tracking in these animals is also devaluation sensitive. To test whether these two types of behaviors rely on similar or different prediction error mechanisms, we next tested whether Kamin blocking effects could be observed across these two classes of behaviors. In Experiment 3 we asked if sign-tracking to a lever CS could block the development of goal-tracking to a tone CS; whereas in Experiment 4, we examined whether goal-tracking to a tone CS could block sign-tracking to a lever CS. In both experiments blocking effects were observed suggesting that both sign- and goal-tracking emerge via a common prediction error mechanism. Collectively, the studies reported here suggest that the psychological mechanisms mediating sign- and goal-tracking are more similar than is commonly acknowledged.

Junk-food enhances conditioned food cup approach to a previously established food cue, but does not alter cue potentiated feeding; implications for the effects of palatable diets on incentive motivation.

Efforts to stem the global rise in obesity have been minimally effective, perhaps in part because our understanding of the psychological and behavioral drivers of obesity is limited. It is well established that stimuli that are paired with palatable foods can powerfully influence food-seeking and feeding behaviors. However, how consumption of sugary, fatty "junk-foods" affects these motivational responses to food cues is poorly understood. Here, we determined the effects of short- and long-term "junk-food" consumption on the expression of cue potentiated feeding and conditioned food cup approach to Pavlovian conditioned stimuli (CS). Further, to determine the degree to which effects of "junk-food" were selective to Pavlovian motivational processes, we varied the predictive validity of the CS by including training groups conditioned with unique CS-US contingencies ranging from -1.0 to +1.0. "Junk-food" did not enhance cue potentiated feeding in any group, but expression of this potentiation effect varied with the CS-US contingency independent of diet. In contrast, "junk-food" consistently enhanced conditioned approach to the food cup; this effect was dependent on the previously established CS-US contingency. That is, consumption of "junk-food" following training enhanced approach to the food cup only in response to CSs with previously positive CS-US contingencies. This was accompanied by reduced motivation for the US itself. Together these data show that "junk-food" consumption selectively enhances incentive motivational responses to previously established food CSs, without altering cue potentiated feeding induced by these same CSs, and in the absence of enhanced motivation for food itself.

Enhanced incentive motivation in obesity-prone rats is mediated by NAc core CP-AMPARs.

Studies in humans suggest that stronger incentive motivational responses to Pavlovian food cues may drive over-consumption leading to and maintaining obesity, particularly in susceptible individuals. However, whether this enhanced incentive motivation emerges as a consequence of obesity or rather precedes obesity is unknown. Moreover, while human imaging studies have provided important information about differences in striatal responsiveness between susceptible and non-susceptible individuals, the neural mechanisms mediating these behavioral differences are unknown. The Nucleus Accumbens (NAc) mediates cue-triggered reward seeking and activity in the NAc is enhanced in obesity-susceptible populations. Therefore here, we used selectively-bred obesity-prone and obesity-resistant rats to examine intrinsic differences in incentive motivation, and the role of NAc AMPARs in the expression of these behaviors prior to obesity. We found that obesity-prone rats exhibit robust cue-triggered food-seeking (Pavlovian-to-instrumental transfer, PIT). Using intra-NAc infusion of AMPAR antagonists, we show that this behavior is selectively mediated by CP-AMPARs in the NAc core. Additionally, biochemical data suggest that this is due in part to experience-induced increases in CP-AMPAR surface expression in the NAc of obesity-prone rats. In contrast, in obesity-resistant rats PIT was weak and unreliable and training did not increase NAc AMPAR surface expression. Collectively, these data show that food cues acquire greater incentive motivational control in obesity-susceptible populations prior to the development of obesity. This provides support to the idea that enhanced intrinsic incentive motivation may be a contributing factor, rather than a consequence of obesity. In addition, these data demonstrate a novel role for experience-induced up-regulation of NAc CP-AMPARs in PIT, pointing to potential mechanistic parallels between the processes leading to addiction and to obesity.

Extinction of specific stimulus-outcome (S-O) associations in Pavlovian learning with an extended CS procedure.

Three experiments with male and female rats were conducted to examine the effects of Pavlovian extinction training on Pavlovian-to-instrumental transfer (PIT) in a task in which the unconditioned stimulus (US) was presented at an early time point within an extended conditioned stimulus (CS). Two instrumental responses were trained with different reinforcing outcomes (R1-O1, R2-O2) and then, independently, 2 stimuli were trained with those outcomes (S1-O1, S2-O2). One group then underwent an extinction treatment (S1-, S2-) and a second was merely exposed to the experimental contexts without any stimulus events. Finally, the effects of the 2 stimuli on instrumental responding were assessed in PIT tests. Across experiments we varied the number of Pavlovian training trials prior to extinction (8, 16, or 64 trials) and the length of time following extinction prior to test (i.e., 1 or 21 days, in a test for spontaneous recovery). We observed that outcome-specific PIT was reduced by extinction in all of our training conditions and that this extinction effect was durable, surviving a 3-week spontaneous recovery interval even though conditioned magazine approach spontaneously recovered over this interval. Although extinction reduced the magnitude of PIT, the temporal expression of PIT was mostly unaffected. We found these effects in both male and female rats, though in 1 study females were extinction-resistant. These data suggest that under the conditions studied here Pavlovian extinction may permanently weaken the ability of Pavlovian cues to retrieve a representation of their associated outcomes without impacting the temporal organization of responding. This suggests that different features of learning may be differentially sensitive to extinction. (PsycINFO Database Record

Structural and Functional Plasticity within the Nucleus Accumbens and Prefrontal Cortex Associated with Time-Dependent Increases in Food Cue-Seeking Behavior.

Urges to consume food can be driven by stimuli in the environment that are associated with previous food experience. Identifying adaptations within brain reward circuits that facilitate cue-induced food seeking is critical for understanding and preventing the overconsumption of food and subsequent weight gain. Utilizing electrophysiological, biochemical, and DiI labeling, we examined functional and structural changes in the nucleus accumbens (NAc) and prefrontal cortex (PFC) associated with time-dependent increases in food craving ('incubation of craving'). Rats self-administered 60% high fat or chow 45 mg pellets and were then tested for incubation of craving either 1 or 30 days after training. High fat was chosen for comparison to determine whether palatability differentially affected incubation and/or plasticity. Rats showed robust incubation of craving for both food rewards, although responding for cues previously associated with high fat was greater than chow at both 1 and 30 days. In addition, previous experience with high-fat consumption reduced dendritic spine density in the PFC at both time points. In contrast, incubation was associated with an increase in NAc spine density and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated transmission at 30 days in both groups. Finally, incubation of craving for chow and high fat was accompanied by an increase in calcium-permeable and calcium-impermeable AMPARs, respectively. Our results suggest that incubation of food craving alters brain reward circuitry and macronutrient composition specifically induces cortical changes in a way that may facilitate maladaptive food-seeking behaviors.

Superior ambiguous occasion setting with visual than temporal feature stimuli.

Three experiments with rats compared the relative ease with which different sets of visual or temporal cues could participate in Pavlovian learning. In Experiment 1, 1 group was trained to discriminate between visual cues (Light vs. Dark), whereas the other group learned to discriminate between temporal cues (early [10 s] vs. late [90 s]). Both groups learned to distinguish food-paired from nonpaired periods equally well. In Experiment 2, 2 groups were trained on an ambiguous occasion setting task. For Group Visual, a 2-min Light period signaled that 1 10-s auditory conditioned stimulus, CS1, was reinforced with 1 unconditioned stimulus, US1, but that CS2 was not reinforced; whereas a 2-min dark period signaled that CS1 was not reinforced, but CS2 was reinforced with US2 (i.e., Light: CS1-US1, CS2-; Dark: CS1-, CS2-US2). For Group Temporal, early (10-s) or late (90-s) temporal cues within each of these Light and Dark periods were diagnostic of these contingencies (i.e., Early: CS1-US1, CS2-; Late: CS1-, CS2-US2). Group Visual learned the task, but Group Temporal did not. In Experiment 3 we demonstrated that animals could not solve a related temporal ambiguous occasion setting task in which 1 visual stimulus signaled that both CSs were reinforced early whereas the other visual stimulus signaled that the CSs were reinforced only late. Contrary to a currently popular information theory approach to timing in Pavlovian learning, these results suggest that overt nontemporal visual stimuli are better incorporated into conditional discrimination learning than are temporal stimuli. (PsycINFO Database Record